Transcription in all cells is mediated by multi-subunit DNA-dependent RNA polymerases that are conserved in sequence, structure and function from bacteria to humans. During transcription initiation, RNA polymerase can engage in "abortive initiation"~tens or hundreds of cycles of synthesis and release of short abortive RNA transcripts (-2-15 nt) prior to promoter escape and entry into transcription elongation. Recent work has shown that abortive RNA transcripts are generated in vivo, and accumulate to detectable levels in vivo, raising the possibility that abortive transcripts can play functional, physiologically important, roles in vivo. The proposed research will employ genetic and biochemical approaches coupled with ultra-high-throughput sequencing to address two aims designed to explore the hypothesis that abortive RNA transcripts play regulatory functions in vivo (in the model organism Escherichia coli). The first aim is to systematically identify abortive RNA transcripts produced in vivo (the "abortome"). The second aim is to determine whether abortive transcripts can act as primers for transcription initiation in vivo or as antisense effectors in vivo. Abortive RNA transcripts are produced by all characterized multisubunit RNA polymerases;bacterial, archaeal, and eukaryotic. Thus, examining whether abortive RNA transcripts can play regulatory roles in vivo has the potential to identify a novel class of regulatory small RNAs that may exist in all domains of life. In this work, we will investigate the possible existence of a novel class of regulatory small RNAs. Because small RNAs have emerged as key regulators of numerous developmental pathways and disease processes, the proposed research may lead to discoveries with important implications for public health.